计算溶液所需的质量、体积或浓度。
磷酸二酯酶 (PDEs) 活性检测试剂盒 (BNPP, 微量法)
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BioReagent,适用于分析 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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2-8°C储存,避光,室温,-20°C储存。低温运输 。请查阅批次 COA 获取详细规格。
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
产品介绍
磷酸二酯酶(phosphodiesterases,PDEs)(EC 31.4.1)是一类磷酸水解酶，可以水解环状腺苷酸单磷酸和环状鸟苷酸单磷酸等。
本试剂盒提供一种简单、灵敏、快速的的检测方法。催化底物双(4-硝基苯)磷酸酯(BNPP)分解生成黄色的产物PNP，该产物在405nm处有最大吸收峰。通过检测PNP在405nm下的增加速率，即可得到磷酸二酯酶(PDEs)活性的大小。
产品组分
组分货号 | 组分名称 | 物理外观 | 48T | 储存 | 单次测试使用量 |
P1528243A | 提取液 | 无色溶液 | 60 mL | 2-8 ℃. | 按需 |
P1528243B | 试剂一 | 无色溶液 | 20 mL | 2-8 ℃. | 140 μL |
P1528243C | 试剂二 | 类白色粉末 | 2*1EA | 2-8 ℃. Store in the dark. | 按需 |
P1528243D | 试剂三 | 无色溶液 | 4 mL | 室温 | 30 μL |
P1528243E | 标准品 (10 mM) | 浅黄色溶液 | 1.5 mL | -20℃. Store in the dark. | 按需 |
注：
1. 每支临用前短暂离心使粉体落入底部，再加0.7mL蒸馏水混匀，现配现用，建议分装，2-8℃一周内用完，-20℃一个月内用完。
2. 整套试剂盒未拆封时于2-8°C 避光保存，自生产之日起有效期 1 年；拆封后各组分稳定性不同，部分组分可转至 -20°C/RT 保存（有效期仍为 1 年）。请严格按照组分表标注温度分类存放，保障试剂性能；使用前将试剂平衡至室温，尽量避免反复冻融。
所需的仪器和用品
酶标仪、96孔板、低温离心机、水浴锅、可调式移液器、研钵、蒸馏水。
使用说明
建议正式实验前选取2个样本做预测定，了解本批样品和实验流程，避免样本和试剂浪费！
1. 样本制备
1.1. 组织样本：取约0.1g组织(水分充足的样本可取0.5g)，加入1mL提取液，进行冰浴匀浆，4℃×12000rpm离心15min, 取上清液待测。注：若增加样本量，可按照组织质量(g)：提取液体积(mL)为1:5~10的比例提取。
1.2. 细菌/细胞样本：先收集细菌或细胞到离心管内，离心后弃上清；取约500万细菌或细胞加入1mL提取液，超声波破碎细菌或细胞(冰浴，功率200W, 超声3s，间隔10s, 重复30次)；12000rpm 4℃离心10min, 取上清，置冰上待测。注：若增加样本量，可按照细菌/细胞数量(10⁴):提取液(mL)为500~1000的比例进行提取。
1.3. 液体样本：可直接测定，或者适当稀释后测定。若浑浊，离心后取上清检测。
2. 上机检测
2.1. 酶标仪预热30min，设置温度37℃，调节波长为405nm。
2.2. 所有试剂于37℃水浴中预热30min。
2.3. 在96孔板中依次加入下列试剂：
试剂名称 (μL) | 测定管 | 对照管 | 空白管(只做一次) |
样本 | 10 | 10 | - |
蒸馏水 | - | 20 | 10 |
试剂一 | 140 | 140 | 140 |
试剂二 | 20 | - | 20 |
混匀，避光反应，37℃水浴或恒温培养箱孵育20min。 | |||
试剂三 | 30 | 30 | 30 |
混匀，在37℃下静置5min，立即于405nm下读取吸光值A，ΔA = A测定 − A对照 − A空白。 |
3. 结果计算
3.1. 标准曲线：y=0.05539x-0.003426；x是PNP 摩尔质量：nmol，y是∆A。
3.2. 按照样本质量计算：
定义：在37℃下，每克组织每分钟水解1nmol的BNPP产生PNP定义为1个酶活单位。
PDEs(nmol/min/g 鲜重)=[(∆A+0.003426)÷0.05539]÷(W×V₁÷V)÷T×D=90.27×(∆A+0.003426)÷W×D
3.3. 按照样本蛋白浓度计算：
定义：在37℃下，每毫克蛋白每分钟水解1nmol的BNPP产生PNP 定义为1个酶活单位。
PDEs(nmol/min/mg prot)=[(∆A+0.003426)÷0.05539]÷(Cₚᵣ×V₁)÷T×D=90.27×(∆A+0.003426)÷Cₚᵣ×D
3.4. 按细菌/细胞数量计算：
定义：在37℃下，每10⁴个细胞每分钟水解1nmol的BNPP产生PNP定义为一个酶活单位。
PDEs(nmol/min/10⁴ cell)=[(∆A+0.003426)÷0.05539]÷(500×V₁÷V)÷T×D=90.27×(∆A+0.003426)÷500×D
3.5. 按液体体积计算：
定义：在37℃下，每毫升液体每分钟水解lnmol的BNPP产生PNP定义为1个酶活单位。
PDEs 活力(nmol/min/mL)=[(∆A+0.003426)÷0.05539]÷V1÷T×D=90.27×(∆A+0.003426)×D
W---样品质量，g；
V---提取液体积，1mL；
V₁---上清液体积(mL)，0.01mL；
T---反应时间，20min。
D---稀释倍数，未稀释即为1；
500---细胞数量，万；
Cₚᵣ---上清液蛋白质浓度，mg/mL；建议使用本公司的BCA 蛋白质含量测定试剂盒。
4. 附：标准曲线制作过程：
4.1. 把标准品(10mM)稀释成以下浓度梯度的标准品：0，0.4，0.8，1.2，1.6，2μmol/mL。也可根据实际样本来调整标准品浓度，0-4μmol/mL仍然成良好的线性关系。
4.2. 依据10μL 标准品+140μL试剂一+20μL蒸馏水+30μL试剂三，混匀，在37℃下静置5min后于405nm下读取吸光值A，根据结果即可制作标准曲线。
注意事项
1. 待测样品中不能含有磷酸酶抑制剂，同时需避免反复冻融。
2. 建议每次测定时都做标准曲线，以使标准更准确，另外标准品避免反复冻融。
3. 检测组织和细胞样品时，需测定总蛋白浓度，推荐使用BCA蛋白定量试剂盒。
4. 配制一支试剂二临用前短暂离心使粉体落入底部，再加0.7mL蒸馏水混匀，现配现用，建议分装，避光保存，2-8℃一周内用完，-20℃一个月内用完。因此请注意适当多准备一些样品一起检测。
5. 标准品溶液对人体有害，试剂三有腐蚀性，请注意个人防护，小心操作。
6. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Introduction
Phosphodiesterases (PDEs) (EC 3.1.4.1) are a class of phosphohydrolases that can hydrolyze cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), etc.
This kit provides a simple, sensitive, and rapid detection method. It catalyzes the decomposition of the substrate bis(4-nitrophenyl) phosphate (BNPP) to produce the yellow product p-nitrophenol (PNP), which has a maximum absorption peak at 405 nm. By measuring the increase rate of PNP at 405 nm, the activity of PDEs can be determined.
Kit Contents
Component No. | Component Name | Appearance | 48T | Storage | Quantity Per Test |
P1528243A | Extraction Solution | Colourless liquid | 60 mL | 2-8 ℃. | As needed |
P1528243B | Reagent 1 | Colourless liquid | 20 mL | 2-8 ℃. | 140 μL |
P1528243C | Reagent 2 | Off-White powder | 2*1EA | 2-8 ℃. Store in the dark. | As needed |
P1528243D | Reagent 3 | Colourless liquid | 4 mL | RT. | 30 μL |
P1528243E | Standard (10 mM) | Light yellow liquid | 1.5 mL | -20℃. Store in the dark. | As needed |
Notes:
1. Before use, briefly centrifuge each vial to pellet the powder at the bottom. Add 0.7 mL distilled water and mix thoroughly. Prepare freshly before use. Aliquoting is recommended. Store in the dark. Store at 2–8 °C and use within one week, or at −20 °C and use within one month.
2. Store the unopened complete kit at 2-8 °C protected from light, with a 1‑year shelf‑life from the date of manufacture. After opening, components differ in stability: some may be transferred to -20 °C/RT (1‑year shelf‑life retained). Store components separately as specified in the component list for optimal performance. Equilibrate reagents to room temperature before use and avoid repeated freeze‑thaw cycles.
Instruments and Materials Required
Microplate reader, 96-well plate, refrigerated centrifuge, water bath, adjustable pipettes, mortar and pestle, distilled water.
Instruction for Use
It is recommended to select 2 samples for a preliminary assay before the formal experiment to understand the sample batch and experimental procedure, and avoid waste of samples and reagents.
1. Sample Preparation
1.1.Tissue samples: Take about 0.1 g tissue (0.5 g may be used for samples with sufficient moisture), add 1 mL Extraction Solution, homogenize in an ice bath, centrifuge at 4°C × 12,000 rpm for 15 min, and take the supernatant for testing. Note: If increasing the sample amount, extract at a ratio of tissue mass (g): extraction solution volume (mL) = 1:5–10.
1.2. Bacteria/cell samples: First collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Take about 5 million bacteria or cells, add 1 mL Extraction Solution, and ultrasonicate to disrupt the bacteria or cells (ice bath, power 200 W, sonication 3 s, interval 10 s, repeat 30 times). Centrifuge at 12,000 rpm at 4°C for 10 min, take the supernatant, and place on ice for testing. Note: If increasing the sample amount, extract at a ratio of bacterial/cell number (10⁴ cells): extraction solution (mL) = 500–1000.
1.3. Liquid samples: Can be assayed directly or after appropriate dilution. If turbid, centrifuge and take the supernatant for detection.
2. Plate Assay
2.1. Preheat the microplate reader for 30 min, set the temperature to 37°C, and adjust the wavelength to 405 nm.
2.2. Preheat all reagents in a 37°C water bath for 30 min.
2.3. Add the following reagents to a 96-well plate in order:
Reagent (μL) | Measurement Well | Control Well | Blank Well (once only) |
Sample | 10 | 10 | - |
Distilled water | - | 20 | 10 |
Reagent 1 | 140 | 140 | 140 |
Reagent 2 | 20 | - | 20 |
Mix, incubate at 37 °C for 20 min in a water bath or incubator, protected from light. | |||
Reagent 3 | 30 | 30 | 30 |
Mix, let stand at 37°C for 5 min, immediately read absorbance A at 405 nm. ΔA = Ameasurement −Acontrol − Ablank |
3. Calculation of Results
3.1. Standard curve: y = 0.05539x − 0.003426; x is the molar amount of PNP: nmol; y is ΔA.
3.2. Calculation based on sample mass:
Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes 1 nmol BNPP to PNP per minute per gram of tissue at 37°C.
PDEs (nmol/min/g tissue) = [(ΔA + 0.003426) ÷ 0.05539] ÷ (W × V₁ ÷ V) ÷ T × D = 90.27 × (ΔA + 0.003426) ÷ W × D
3.3. Calculation based on sample protein concentration:
Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes 1 nmol BNPP to PNP per minute per mg protein at 37°C.
PDEs (nmol/min/mg prot) = [(ΔA + 0.003426) ÷ 0.05539] ÷ ( Cₚᵣ × V₁) ÷ T × D = 90.27 × (ΔA + 0.003426) ÷ Cₚᵣ × D
3.4. Calculation based on bacterial/cell number:
Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes 1 nmol BNPP to PNP per minute per 10⁴ cells at 37°C.
PDEs (nmol/min/10⁴ cells) = [(ΔA + 0.003426) ÷ 0.05539] ÷ (500 × V₁ ÷ V) ÷ T × D = 90.27 × (ΔA + 0.003426) ÷ 500 × D
3.5. Calculation based on liquid volume:
Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes 1 nmol BNPP to PNP per minute per mL liquid at 37°C.
PDEs activity (nmol/min/mL) = [(ΔA + 0.003426) ÷ 0.05539] ÷ V₁ ÷ T × D = 90.27 × (ΔA + 0.003426) × D
Where:
W --- sample mass, g;
V --- extraction solution volume, 1 mL;
V₁ --- supernatant volume (mL), 0.01 mL;
T --- reaction time, 20 min;
D --- dilution factor; 1 if undiluted;
500 --- cell number, in ten thousands (i.e., 5,000,000 cells);
Cₚᵣ --- protein concentration of supernatant, mg/mL; the company's BCA Protein Assay Kit is recommended.
4. Appendix: Standard Curve Preparation
4.1. Dilute the Standard (10 mM) to the following concentration gradient: 0, 0.4, 0.8, 1.2, 1.6, 2 μmol/mL. The standard concentrations can also be adjusted according to actual samples. A good linear relationship was maintained in the range of 0–4 μmol/mL.
4.2. Use 10 μL standard + 140 μL Reagent 1 + 20 μL distilled water + 30 μL Reagent 3, mix well, let stand at 37°C for 5 min, then read absorbance A at 405 nm. The standard curve can be prepared from the results.
Precautions
1. Avoid repeated freeze thaw cycles of the His tagged ubiquitin binding protein. Aliquot upon first use as needed. Samples to be tested must not contain phosphatase inhibitors, and repeated freeze-thaw cycles should be avoided.
2. It is recommended to prepare a standard curve for each assay to make the standard more accurate. Avoid repeated freeze-thaw cycles of the standard.
3. When detecting tissue and cell samples, total protein concentration must be determined. BCA Protein Assay Kit is recommended.
4. Before use, briefly centrifuge one vial of Reagent 2 to collect the powder at the bottom, add 0.7 mL distilled water, and mix well. Prepare fresh before use; aliquoting is recommended. Use within 1 week at 2–8°C or within 1 month at -20°C. Therefore, please prepare an appropriate number of samples for testing together.
5. The standard solution is harmful to humans, and Reagent 3 is corrosive. Please take personal protection and operate carefully.
6. For your safety and health, please wear a lab coat and disposable gloves.
规格
规格或纯度
BioReagent, 适用于分析
英文名称
Phosphodiesterase (PDEs) Activity Assay Kit (BNPP, Micro Method)
应用
Biochemical assay, 细胞代谢, 酶活性检测
样品类型
组织, 细胞, 细菌, 血浆, 血清
检测范围
4-40 nmol
检测方法
微量法
定量&半定量
定量
检测仪器
酶标仪
检测波长
405 nm
敏感性
Light-sensitive
检测时长
0.5 h
样本体积
10 μL
储存与运输
储存条件
2-8°C储存,避光,室温,-20°C储存
运输条件
低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年。
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 48T | 储存 | 单次测试使用量 |
| P1528243A | 提取液 | 无色溶液 | 60 mL | 2-8 ℃. | 按需 |
| P1528243B | 试剂一 | 无色溶液 | 20 mL | 2-8 ℃. | 140 μL |
| P1528243C | 试剂二 | 类白色粉末 | 2*1EA | 2-8 ℃.避光 | 按需 |
| P1528243D | 试剂三 | 无色溶液 | 4 mL | 室温 | 30 μL |
| P1528243E | 标准品 (10 mM) | 浅黄色溶液 | 1.5 mL | -20℃. 避光 | 按需 |
注：
1. 每支临用前短暂离心使粉体落入底部，再加0.7mL蒸馏水混匀，现配现用，建议分装，避光保存，2-8℃一周内用完，-20℃一个月内用完。
2. 整套试剂盒未拆封时于2-8°C 避光保存，自生产之日起有效期 1 年；拆封后各组分稳定性不同，部分组分可转至 -20°C/RT 保存（有效期仍为 1 年）。请严格按照组分表标注温度分类存放，保障试剂性能；使用前将试剂平衡至室温，尽量避免反复冻融。
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